Electrochemical cell

ABSTRACT

Devices and methods are provided for determining the concentration of a reduced form of a redox species. For example, a device can include a working electrode and a counter electrode spaced by a predetermined distance so that reaction produces from the counter electrode arrive at the working electrode. An electric potential difference can be applied between the electrodes, and the potential of the working electrode can be selected such that the rate of electro-oxidation of the reduced form of the species is diffusion controlled. Current as a function of time can be determined, the magnitude of the steady state current can be estimated, and a value indicative of the diffusion coefficient and/or of the concentration of the reduced form of the species can be obtained from the change in current with time and the magnitude of the steady state current. Other embodiments of apparatuses, devices, and methods are also provided.

FIELD OF THE INVENTION

This invention relates to a biosensor and more particularly to anelectrochemical biosensor for determining the concentration of ananalyte in a carrier. The invention is particularly useful fordetermining the concentration of glucose in blood and is describedherein with reference to that use but it should be understood that theinvention is applicable to other analytic determinations.

BACKGROUND OF THE INVENTION

Electrochemical biosensors generally comprise a cell having a workingelectrode, a counter electrode and a reference electrode. Sometimes thefunction of the counter and reference electrodes are combined in asingle electrode called a “counter/reference” electrode or “pseudoreference electrode”. As herein used the term “counter electrode”includes a counter/reference electrode where the context so admits.

The sample containing the analyte is brought into contact with a reagentcontaining an enzyme and a redox mediator in the cell. Either themediator is reduced (receives at least one electron) while the analyteis oxidised (donates at least one electron) or visa versa. Usually it isthe analyte which is oxidised and the mediator which is reduced. Theinvention will be herein described principally with reference to thatsystem but it is also applicable to systems in which the analyte isreduced and the mediator oxidised.

Electrochemical glucose analysers such as those used by diabetics tomonitor blood glucose levels or such as are used in clinics andhospitals are commonly based upon the use of an enzyme such as glucoseoxidase dehydrogenase (GOD) and a redox mediator such as a ferricyanideor ferrocyanide. In such prior art system, the sample (e.g. blood)containing the analyte (e.g. glucose) is brought into contact with thereagents in the cell. Glucose is oxidised to gluconic acid and theglucose oxidase is thereby reduced. The mediator then re-oxidizes theglucose oxidase and is reduced in the process. The reduced mediator isthen re-oxidized when it transfers electrons to the working electrode.After allowing passage of a predetermined time, sufficient to obtain anaccurate estimate of the Faraday current, the concentration of glucoseis estimated from the magnitude of the current or voltage signal thenmeasured.

Prior art electrochemical cells consist of two (or three) adjacentelectrodes spaced apart on one side of an insulator and adapted forconnection to a measuring device. A target area on which the bipodsample is placed is defined on or between the electrodes. Co-pendingApplication PCT/AU95/00207 describes a cell in which electrodes aredisposed on opposite sides of a porous membrane, one of the electrodeshaving a liquid permeable target area.

In the prior art there is a need to separate the working electrode fromthe counter (or counter/reference) electrode by a sufficient distance toavoid products of electrochemical reaction at one electrode frominterfering with those at the other. In practice a separation of theelectrodes of more than 500 μm is required to achieve acceptableaccuracy.

Each batch of cells is required to have been previously calibrated andleads to inaccuracies during use because of variations within the batch,in sample composition, and in ambient conditions.

It is desired to improve, the accuracy and reliability of suchbiosensors. Achievement of these objectives is made difficult in thecase of sensors intended to determine the concentration of analytes inblood because blood contains dissolved gases, ions, colloids, complexmicelles, small scale cellular debris, and living cellular components ina predominantly aqueous medium. Any of these may interfere in thedetermination. Existing sensors are also susceptible to influence fromother interfering substances that may be present in the sample and whichmay be oxidised at the working electrode and mistakenly identified asthe analyte of interest. Alternatively, the interfering substances mayreduce the oxidised form of the redox mediator. These effects will giveartificially elevated estimates of the analyte concentration.Additionally there is always some reduced redox mediator present beforethe analyte is added and its concentration needs to be known andsubtracted from the measured value of reduced mediator to give anaccurate concentration of the analyte. Moreover, oxygen in the blood mayact as a redox mediator for glucose oxidase dehydrogenase (GOD) incompetition with ferrocyanide. Thus high oxygen concentrations can leadto low estimates of glucose concentration. In addition the measurementsare sensitive to factors such as changes in humidity, temperature,solution viscosity and haematocrit content.

OBJECT OF THE INVENTION

It is an object of the present invention to provide a method of analysisand apparatus for use in the method which avoid or ameliorate at leastsome of the disadvantages of the prior art It is an object of preferredforms of the invention to provide a biosensor of improved accuracy,and/or reliability and/or speed and a method for its use.

DISCLOSURE OF THE INVENTION

According to one aspect the invention consists in a method fordetermining the concentration of a reduced (or oxidised) form of a redoxspecies in an electrochemical cell of the kind comprising a workingelectrode and a counter electrode spaced from the working electrode by apredetermined distance, said method comprising the steps of:

(1) applying an electric potential difference between the electrodes,

(2) selecting the potential of the working electrode such that the rateof electro-oxidation of the reduced form (or electro-reduction of theoxidised form) of the species is diffusion controlled,

(3) selecting the spacing between the working electrode and the counterelectrode so that reaction products from the counter electrode arrive atthe working electrode,

(4) determining current as a function of time after application of thepotential and prior to achievement of a steady state,

(5) estimating the magnitude of the steady state current, and

(6) obtaining from the change in current with time and the magnitude ofth steady state current, a value indicative of the diffusion coefficientand/or of the concentration of the reduced form (or the oxidised form)of the species.

The concentration measured in this way is substantially independent ofvariation if any in the diffusion coefficient of the reduced form, andtherefore is compensated for variations in temperature and viscosity.The concentration so measured is independent of variations inhaematocrit and other substances which affect the diffusion coefficientof the reduced form of the redox species.

It will be appreciated that the method; of the invention is equallyapplicable for determining the concentration of a reduced form of aredox species or an oxidized form of a redox species in the cell. In thecase that the concentration of the reduced form is to be determined thepotential of the working electrode must be maintained such that the rateof electro oxidation of the reduced form is diffusion controlled in step(2) and it is the concentration of the reduced form that is obtained instep (5). In the case that the concentration of oxidized form is to bedetermined, the potential of the working electrode must be maintainedsuch that the rate of electro reduction of the oxidized form isdiffusion controlled in step (2) and it is the concentration of theoxidized form that is obtained in step (5).

The redox species may be an analyte or may be a redox mediator.

In preferred embodiments of the method a mediator is used and theconcentration of the reduced (or oxidized) form of the mediator is inturn indicative of the concentration of an analyte and a measure of thediffusion coefficient of the reduced (or oxidized) form of the mediatoris determined as a precursor to the determination of the concentrationof the analyte.

For preference the cell comprises a working electrode andcounter/reference electrode. If a reference electrode separate from acounter electrode is used, then the reference electrode may be in anyconvenient location in which it is in contact with the sample in thesensor.

In contrast to prior art, when conducting the method of the invention,the electrodes arc sufficiently close that the products ofelectrochemical reaction at the counter electrode migrate to the workingelectrode during the period of the test. For example, in an enzymeferricyanide system, the ferrocyanide produced at the counter electrodediffuses to the working electrode.

This allows a steady state concentration profile to be achieved betweenthe electrodes leading to a steady state current. This in turn allowsthe diffusion coefficient and concentration of the redox species(mediator) to be measured independently of sample variations andtherefore greatly improves accuracy and reliability.

The method also permits the haematocrit concentration of blood to bedetermined from the diffusion coefficient by use of look-up tables (orby separation of red cells from plasma and measurement of the diffusioncoefficient of the red cell fraction) and the plasma fraction, andcomparing the two.

According to a second aspect, the invention consists in apparatus fordetermining the concentration of a redox species in an electrochemicalcell comprising:

an electrochemical-cell having a working electrode and a counter (orcounter/reference) electrode,

means for applying an electric potential difference between saidelectrodes, means for measuring the change in current with time,

and characterised in that the working electrode is spaced from thecounter electrode by less than 500 μm.

In preferred embodiments the cell has an effective volume of 1.5microlitres or less. Apparatus for use in the invention may comprise aporous membrane, a working electrode on one side of the membrane, acounter/reference electrode on the other side, said electrodes togetherwith a zone of the membrane therebetween defining an electrochemicalcell, and wherein the membrane extends laterally from the cell to asample deposition area spaced apart from the cell zone by a distancegreater than the thickness of the membrane.

Preferably the porous membrane, the distance of the target area from thecell portion, and the membrane thickness are so selected in combinationthat when blood (comprising plasma and red cells) is placed on thetarget area a plasma front diffuses laterally towards theelectrochemical cell zone in advance of the red cells.

It is thus possible to fill a thin layer electrochemical cell withplasma substantially free of haematocrit which would cause a variationin the diffusion coefficient of the redox mediator and which wouldaffect the accuracy of the test as hereinafter explained.

In preferred embodiments of the biosensor according to the invention asecond electrochemical cell zone of the membrane is defined by a secondworking electrode and a second counter/reference electrode on theopposite side of the membrane from the second working electrode. Thesecond electrochemical cell zone is situated intermediate the first cellzone and the sample deposition or “target” area, or is situated on theside of the target area remote from the first electrochemical zone. Inthese embodiments the plasma comes into contact with enzyme in, or onroute to, the first electrochemical cell while plasma reaching thesecond cell does not. The first cell thus in use measures theconcentration of reduced mediator in the presence of plasma(includingelectrochemically interfering substances), and enzyme while the secondelectrochemical cell measures it in the presence of plasma (includingelectrochemically interfering substances) and in the absence of enzyme.This allows determination of the concentration of the reducedinterfering substances in the second cell and the concentration ofreduced interfering substances plus analyte in the first cell.Subtraction of the one value from the other gives the absoluteconcentration of analyte.

In a highly preferred embodiment of the invention a hollow cell isemployed wherein the working and reference (or counter/reference)electrodes are spaced apart by less than 500 μμm and preferably by from20-200 μm.

DESCRIPTION OF THE DRAWINGS

The invention will now be more particularly described by way of exampleonly with reference to the accompanying drawings wherein:

FIG. 1 is a schematic drawing (not to scale) of a first embodiment,according to the invention shown in side elevation.

FIG. 2 shows the embodiment of FIG. 1 in plan, viewed from above.

FIG. 3 shows the embodiment of FIG. 1 in plan, viewed from below.

FIG. 4 shows the embodiment of FIG. 1 viewed in end elevation.

FIG. 5 is a schematic drawing (not to scale) of a second embodimentaccording to the invention in side elevation.

FIG. 6 shows the embodiment of FIG. 5 in plan, viewed from above.

FIG. 7 is a schematic drawing (not to scale) of a third embodimentaccording to the invention, in side elevation.

FIG. 8 shows the embodiment of FIG. 7 in plan, viewed from above.

FIG. 9 is a schematic drawing (not to scale) according to the inventionin plan view, viewed from above.

FIG. 10 shows the embodiment of FIG. 9 in end elevation.

FIG. 11 shows me embodiment of FIG. 9 in side elevation.

FIG. 12 shows a schematic drawing (not to scale) of a hollow cellembodiment according to the invention, viewed in cross section.

FIG. 13 is a graph showing a plot of current (ordinate axis) versus time(co-ordinate axis) during conduct of a method according to theinvention.

FIG. 14 is a further graph of use in explaining the method of theinvention.

In FIGS. 5 to 12, components corresponding in function to components ofthe embodiment of FIGS. 1 to 4 are identified by identical numerals orindicia.

DESCRIPTION OF PREFERRED EMBODIMENTS

With reference to FIGS. 1 to 4 there is shown a first embodiment ofapparatus of the invention, in this case a biosensor for determiningglucose in blood. The embodiment comprises a thin strip membrane 1having upper and lower surfaces 2, 3 and having a cell zone 4 definedbetween a working electrode 5 disposed on upper surface 2 and a counterelectrode 6 disposed on lower surface 3. The membrane thickness isselected so that the electrodes are separated by a distance “l” which issufficiently close that the products of electrochemical reaction at thcounter electrode migrate to the working electrode during the time ofthe test and a steady state diffusion profile is substantially achieved.Typically, “l” will be less than 500 μm. A sample deposition or “target”area 7 defined on upper surface 2 of membrane 1 is spaced at a distancegreater than the membrane thickness from cell zone 4. Membrane 1 has adiffusion zone 8 extending between target area 7 and cell zone 4. Asuitable reagent including a redox mediator “M”, an enzyme “E” and a pHbuffer “B” are contained within cell zone 4 of the membrane and/orbetween cell zone 4 and target area 7. The reagent may also includestabilisers and the like.

In some cases it is preferable to locate the enzyme and mediator and/orthe buffer in different zones of the membrane. For example the mediatormay be initially located within electrochemical cell zone 4 while theenzyme may be situated below target area 7 or in diffusion zone 8.

Haemoglobin releases oxygen at low pH's, but at higher pH's it bindsoxygen very firmly. Oxygen acts as a redox mediator for glucose oxidasedehydrogenase (GOD). In a glucose sensor this competes with the redoxmediator leading to low estimates of glucose concentration. Therefore ifdesired a first pH buffer can be contained in the vicinity of targetarea 7 to raise the pH to such a level that all the oxygen is bound tohaemoglobin. Such a pH would be non-optimal for GOD/glucose kinetics andwould consequently be detrimental to the speed and sensitivity of thetest. In a preferred embodiment of the invention a second pH buffer iscontained as a reagent in the vicinity of the working electrode torestore the pH to kinetically optimal levels. The use of a second bufferdoes not cause oxygen to be released from the haemoglobin as thehaemoglobin is contained within the blood cells which are retained nearblood target area 7 or are retarded in diffusion in comparison with theplasma and therefore not influenced by the second buffer. In this manneroxygen interference may be greatly reduced or eliminated.

In use of the sensor a drop of blood containing a concentration ofglucose to be determined is placed on target zone 7. The bloodcomponents wick towards cell zone 4, the plasma component diffusing morerapidly than red blood cells so that a plasma front reaches cell zone 4in advance of blood cells.

When the plasma wicks into contact with the reagent, the reagent isdissolved and a reaction occurs that oxidises the analyte and reducesthe mediator. After allowing a predetermined time to complete thisreaction an electric potential difference is applied between the workingelectrode and the counter electrode. The potential of the workingelectrode is kept sufficiently anodic such that the rate of electrooxidation of the reduced form of the mediator at the working electrodeis determined by me rate of diffusion of the reduced form of themediator to the working electrode, and not by the rate of electrontransfer across the electrode/solution interface.

In addition the concentration of the oxidised form of the mediator atthe counter electrode is maintained at a level sufficient to ensure thatwhen a current flows in the electrochemical cell the potential of thecounter electrode, and thus also the potential of the working electrode,is not shifted so far in the cathodic direction that the potential ofthe working electrode is no longer in the diffusion controlled region.That is to say, the concentration of the oxidized form at the counterelectrode must be sufficient to maintain diffusion controlled electrooxidation of the reduced form of the mediator at the working electrode.

The behaviour of a thin layer cell is such that if both oxidised andreduced forms of the redox couple are present, eventually a steady stateconcentration profile is established across the cell. This results in asteady state current. It has been found that by comparing a measure ofthe steady state current with the rate at which the current varies inthe current transient before the steady state is achieved, the diffusioncoefficient of the redox mediator can be measured as well as itsconcentration. This is in contrast to the Cottrell current that ismeasured in the prior art. By measuring the Cottrell current at knowntimes after application of a potential to the sensor electrodes it isonly possible to determine the product concentration times square rootof the diffusion coefficient and therefore it is not possible todetermine the concentration of the mediator independent of its diffusioncoefficient.

In a cell according to the current invention, by solving the appropriatediffusion equations it can be shown that over a restricted time range aplot of ln(i/i^(∞)−1) vs time (measured in seconds) is linear and has aslope (denoted by S) which is equal to −4π²D/l², where “i” is thecurrent at time “t”, “i^(∞)” is the steady state current, “D” is thediffusion. coefficient in cm²/sec, “l” is the distance between theelectrodes in cm and “π” is approximately 3.14159. The concentration ofreduced mediator present when the potential was applied between theelectrodes is given by 2π²i^(∞)FAlS, where “F” is Faraday's constant, Ais the working electrode area and the other symbols are as given above.As this later formula uses S it includes the measured value of thediffusion coefficient.

Since l is a constant for a given cell, measurement of i as a functionof time and i^(∞) enable the value of the diffusion coefficient of theredox mediator to be calculated and the concentration of the analyte tobe determined.

Moreover the determination of analyte concentration compensates for anyvariation to the diffusion coefficient of the species which is electrooxidised or electro reduced at the working electrode. Changes in thevalue of the diffusion coefficient may occur as a result of changes inthe temperature and viscosity of the solution or variation of themembrane permeability. Other adjustments to the measured value of theconcentration may be necessary to account for other factors such aschanges to the cell geometry, changes to the enzyme chemistry or otherfactors which may effect the measured concentration. If the measurementis made oh plasma substantially free of hematocrit (which if presentcauses variation in the diffusion coefficient of the redox mediator) theaccuracy of the method is further improved.

Each of electrodes 5, 6 has a predefined area. In the embodiments, ofFIGS. 1 to 4 cell zone 4 is defined by edges 9, 10, 11 of the membranewhich correspond with edges of electrodes 5, 6 and by leading (withrespect to target area 7) edges 12, 13 of the electrodes. In the presentexample the electrodes are about 600 angstrom thick and are from 1 to 5mm wide.

Optionally, both sides of the membrane are covered with the exception ofthe target area 7 by laminating layers 14 (omitted from plan views)which serves to prevent evaporation of water from the sample and toprovide mechanical robustness to the apparatus. Evaporation of water isundesirable as it concentrates the sample, allows the electrodes to dryout, and allows the solution to cool, affecting the diffusioncoefficient and slowing the enzyme kinetics, although diffusioncoefficient can be estimated as. above.

A second embodiment according to the invention, shown in FIGS. 5 and 6,differs from the first embodiment by inclusion of a second workingelectrode 25 and counter/reference electrode 26 defining a second cellzone 24 therebetween. These electrodes are also spaced apart by lessthan 500 μm in the present example. Second electrodes 25, 26 axesituated intermediate cell zone 4 and target area 7. In this embodimentthe redox mediator is contained in the membrane below or adjacent totarget area 7 or intermediate target area 7 and first cell zone 4. Theenzyme is contained in the membrane in the first cell zone 4 and secondcell zone 24. The enzyme does not extend into second cell 24. In thiscase when blood is added to the target area, it dissolves the redoxmediator. This wicks along the membrane so that second electrochemicalcell 24 contains redox mediator analyte and serum includingelectrochemically interfering substances. First electrochemical cellreceives mediator, analyte, serum containing electrochemicallyinterfering substances, and enzyme. Potential is now applied betweenboth working electrodes and the counter electrode or electrodes but thechange in current with time is measured separately for each pair. Thisallows the determination of the concentration of reduced mediator in theabsence of analyte plus the concentration of electrochemicallyinterfering substances in the second electrochemical cell and theconcentration of these plus analyte in me first electrochemical cell.Subtraction of the one value from the other gives the absoluteconcentration of analyte.

The same benefit is achieved by a different geometry in the embodimentof FIGS. 7 and 8 in which the second working electrode and secondcounter/reference electrode define me second cell 24 on me side oftarget area 7 remote from first electrochemical cell 4. In this case theenzyme may be contained in the membrane strip between the target areaand cell 1. The redox mediator may be in the vicinity of the target areaor between the target area and each cell. The diffusion coefficient ofmediator is lowered by undissolved enzyme and the arrangement of FIGS. 7and 8 has the advantage of keeping enzyme put of the thin layer cellsand allowing a faster test (as the steady state current is reached morequickly). Furthermore the diffusion constant of redox mediator is thenthe same in both thin layer cells allowing more accurate subtraction ofinterference.

Although the embodiments of FIGS. 1 to 8 are unitary sensors, it will beunderstood that a plurality of sensors may be formed on a singlemembrane as shown in the embodiment of FIGS. 9 to 11. In this case theelectrodes of one sensor are conductively connected to those of anadjacent sensor. Sensors may be used successively and severed from thestrip after use.

In the embodiment of FIGS. 9 to 11 electrode dimensions are defined inthe diffusion direction (indicated by arrow) by the width of theelectrode in that direction. The effective dimension of the electrode ina direction transverse to diffusion direction is defined between-compressed volumes 16 of the membrane in a manner more fully describedin co-pending Application PCT/AU96/00210, the disclosure of which isincorporated herein by reference in its entirety. For clarity optionallaminated layer 14 of FIG. 1 has been omitted from FIGS. 9 to 11.

In the embodiment of FIG. 12 there is shown a hollow cell according tothe invention wherein the electrodes 5,6 are supported by spaced apartpolymer walls 30 to define a hollow cell. An opening 31 is provided onone side of the cell whereby a sample can be admitted into cavity 32. Inthis embodiment a membrane is not used. As in previous embodiments, theelectrodes are spaced apart by less than 500 μm, preferably 20-400 μmand more preferably 20-200 μm. Desirably the effective cell volume is1.5 microlitres or less.

It will be understood that the method of the invention may be performedwith a cell constructed in accord with co-pending applicationPCT/AU95/00207 or cells of other known design, provided these aremodified to provide a sufficiently small distance between electrodefaces.

The method of the invention will now be further exemplified withreference to FIGS. 13 and 14.

EXAMPLE 1

A membrane 130 microns thick was coated on both sides with a layer ofPlatinum 60 nanometers thick. An area of 12.6 sq. mm was defined byimpressing the membrane. 1.5 microlitres of a solution containing 0.2Molar potassium ferricyanide and 1% by weight glucose oxidasedehydrogenase was added to the defined area of the membrane and thewater allowed to evaporate.

The platinum layers were then connected to a potentiostat to be used asthe working and counter/reference electrodes. 3.0 microlitres of anaqueous solution containing 5 millimolar D-glucose and 0.9 wt % NaCl wasdropped on to the defined area of the membrane. After an elapse of 20seconds a voltage of 300 millivolts was applied between the working andcounter/reference electrodes and the current recorded for a further 30seconds at intervals of 0.1 seconds.

FIG. 13 is a graph of current versus time based on the abovemeasurements. Using a value of the steady state current of 26.9microamps the function ln(i/26.9−1) was computed and plotted versustime. The slope of the graph (FIG. 14) is −0.342 which corresponds to adiffusion coefficient of 1.5×10⁻⁶ cm² per second and a corrected glucoseconcentration (subtracting background ferrocyanide) of 5.0 millimolar.

The steady state current is one in which no further significant currentchange occurs during the test As will be understood by those skilled inthe art, a minimum current may be reached after which there may be adrift due to factors such as lateral diffusion, evaporation, interferingelectrochemical reactions or the like. However, in practice it is notdifficult to estimate the “steady state” current (i^(∞)). One method fordoing so involves approximating an initial value for i^(∞). Using thefit of the i versus t data to the theoretical curve a better estimate ofi^(∞) is then obtained. This is repeated reiteratively until themeasured value and approximated value converge to within an acceptabledifference, thus yielding an estimated i^(∞).

In practice, the measurements of current i at time t are made between aminimum time t min and a maximum time t max after the potential isapplied. The minimum and maximum time are determined by theapplicability of the equations and can readily be determined byexperiment of a routine nature. If desired the test may be repeated byswitching off the voltage and allowing the concentration profiles of theredox species to return towards their initial states.

It is to be understood that the analysis of the current v. time curve toobtain values of the Diffusion Co-efficient and/or concentration is notlimited to the method given above but could also be achieved by othermethods.

For instance, the early part of the current v. time curve could beanalysed by the Cottrell equation to obtain a value of D^(1/2)×Co(Co=Concentration of analyte) and the steady state current analysed toobtain a value of D×Co. These 2 values can then be compared to obtain Dand C separately.

It will be understood that in practice of the invention an electricalsignal is issued by the apparatus which is indicative of change incurrent with time. The signal may be an analogue or digital signal ormay be a series of signals issued at predetermined time intervals. Thesesignals may be processed by means of a microprocessor or otherconventional circuit to perform the required calculations in accordancewith stored algorithms to yield an output signal indicative of thediffusion coefficient, analyte concentration, haematocrit concentrationor the like respectively. One or more such output signals may bedisplayed by means of an analogue or digital display.

It is also possible by suitable cell design to operate the cell, as adepletion cell measuring the current required to deplete the mediator.For example in the embodiment of FIG. 5 the method of the invention maybe performed using electrodes 5, 6, which are spaced apart by less than500 μm. An amperometric or voltametric depletion measurement may be madeusing electrodes 5 and 26 which are spaced apart more than 500 μm andsuch that there is no interference between the redox species beingamperometrically determined at electrodes 5, 26.

The depletion measurement may be made prior to, during or subsequent to,the measurement of diffusion coefficient by the method of the invention.This enables a substantial improvement in accuracy and reproducibilityto be obtained.

In the embodiments described the membrane is preferably an asymmetricporous membrane of the kind described in U.S. Pat. No. 4,629,563 andU.S. Pat. No. 4,774,039 both of which are incorporated herein in theirentirety by reference. However symmetrical porous membranes may beemployed. The membrane may be in the form of a sheet, tube, hollow fibreor other suitable form.

If the membrane is asymmetric the target area is preferably on the moreopen side of the asymmetric membrane. The uncompressed membranedesirably has a thickness of from 20 to 500 μm. The minimum thickness isselected having regard to speed, sensitivity, accuracy and cost. Ifdesired a gel may be employed to separate hematocrit from GOD. The gelmay be present between the electrodes and/or in the space between thesample application area and the electrodes.

The working electrode is of any suitable metal for example gold, silver,platinum, palladium, iridium, lead, a suitable alloy. The workingelectrode may be preformed or formed in situ by any suitable method forexample sputtering, evaporation under partial vacuum, by electrodelessplating, electroplating, or the like. Suitable non-metal conductors mayalso be used for electrode construction. For example, conductingpolymers such as poly(pyrrole), poly(aniline), porphyrin “wires”;poly(isoprene) and poly (cis-butadiene) doped with iodine and “ladderpolymers”. Other non-metal electrodes may be graphite or carbon mixedwith a binder, or a carbon filled plastic. Inorganic electrodes such asIn₂O₃ or SnO₂ may also be used. The counter/reference electrode may forexample be of similar construction to the working electrode. Nickelhydroxide or a silver halide may also be used to form thecounter/reference electrode. Silver chloride may be employed but it willbe understood that chloridisation may not be necessary and silver may beused if sufficient chloride ions are present in the blood sample.Although in the embodiments described the working electrode is shown onthe upper surface of the biosensor and the counter/reference electrodeis on the lower surface, these may be reversed.

It is preferable that the working electrode and counter (orcounter/reference) electrodes are of substantially the same effectivegeometric area.

If a separate reference and counter electrode are employed, they may beof similar construction. The reference electrode can be in any suitablelocation.

It will be understood that the features of one embodiment hereindescribed may be combined with those of another. The invention is notlimited to use with any particular combination of enzyme and mediatorand combinations such as are described in EP 0351892 or elsewhere may beemployed. The system may be used to determine analytes other thanglucose (for example, cholesterol) by suitable adaptation of reagentsand by appropriate membrane selection. The system may also be adaptedfor use with media other than blood. For example the method may beemployed to determine the concentration of contaminants such aschlorine, iron, lead, cadmium, copper, etc., in water.

Although the cells herein described have generally planar and parallelelectrodes it will be understood that other configurations may beemployed, for example one electrode could be a rod or needle and meother a concentric sleeve.

It will be apparent to those skilled in the art from the disclosurehereof the invention may be embodied in other forms without departingfrom the inventive concept herein disclosed.

1-30. (canceled)
 31. A hollow electrochemical cell comprising: a workingelectrode and a counter electrode at least partially facing each otherin a diffusion zone, at least one additional electrode disposed in thediffusion zone and upstream of the working electrode and the counterelectrode with respect to a sample flow through the diffusion zone. 32.The hollow electrochemical cell of claim 31, wherein the additionalelectrode is co-planar with the counter electrode.
 33. The hollowelectrochemical cell of claim 32, wherein the additional electrode is anadditional working electrode.
 34. The hollow electrochemical cell ofclaim 31, wherein the additional electrode is co-planar with the workingelectrode.
 35. The hollow electrochemical cell of claim 34, wherein theadditional electrode is an additional working electrode.
 36. A hollowelectrochemical cell comprising: a working electrode and a counterelectrode at least partially facing each other in a diffusion zone, twoadditional electrodes disposed in the diffusion zone and predetermineddistance from the working electrode and the counter electrode, and atleast one of the two additional electrodes being disposed upstream ofthe working electrode and the counter electrode with respect to a sampleflow through the diffusion zone.
 37. The hollow electrochemical cell ofclaim 36, wherein the predetermined distance is less than 500 μm.